Association of Prenatal Exposure to Ambient Air Pollution With Circulating Histone Levels in Maternal Cord Blood

IMPORTANCE Exposure to ambient air pollution has been associated with the risk of carcinogenesis in later life. Changes in histone modifications might have long-term adverse health effects. OBJECTIVE To investigate the association of prenatal exposure to ambient air pollution with levels of circulating total histone H3 and specific trimethylation marks (ie, H3 lysine 4, H3 lysine 36) in maternal cord blood. DESIGN, SETTING, AND PARTICIPANTS The Environmental Influence on Aging (ENVIR ON AGE) birth cohort study included 609 mothers and their newborns. Participants were recruited when mothers entered the Hospital East Limburg (Genk, Belgium) for delivery between February 2010 and January 2017. The inclusion criteria were singleton pregnancies and the ability to fill out questionnaires in Dutch. Data analysis was conducted from March to August 2019. EXPOSURES Exposure to particulate matter with a diameter less than 2.5 μm (PM 2.5 ), black carbon, and nitrogen dioxide during pregnancy was modeled with a high-resolution air pollution model on the basis of maternal address for each trimester of pregnancy as well as for the entire pregnancy. MAIN OUTCOMES AND MEASURES Circulating total histone H3 levels and specific trimethylation marks (ie, trimethylated H3 lysine 4 and trimethylated H3 lysine 36) in cord blood.


Introduction
The fetus is particularly vulnerable to adverse effects of environmental exposures, given that it is growing and developing at a rapid pace. 1 Epidemiologic studies have shown that exposure to airborne particulate matter (PM) during gestation increases the risk of low birth weight and preterm birth. 2 The developmental origin of health and disease concept explains how challenges early in life can affect lifelong health. 3 Determining which molecular mechanisms underlie prenatal exposure to air pollution is important for the discovery of potential novel biomarkers of exposure as well as for the identification of potential mediators between exposure and disease.
Air pollution is known to, at least in part, exert its effects on human health by upregulating oxidative stress and pro-inflammatory responses. 4 Epigenetic mechanisms are involved in the regulation of oxidative stress as well as in inflammatory responses. 5 Histones are small, abundant proteins that help form nucleosomes. Circulating histones can either be secreted from living or apoptotic cells. When released in circulation, they mediate the inflammatory response and can lead to endothelial dysfunction and organ failure. 6 Histone posttranslational modifications (PTMs) are an epigenetic trait induced by oxidative stress 7 and specific inflammatory mediators. 8 Posttranslational modifications have a critical role in the regulation of nucleosome dynamics and the processes of DNA transcription, replication, and repair. 9 Histones have also been shown to have toxic and pro-inflammatory activities when they are released into the extracellular space. 6 We hypothesized that histone modifications may serve as a molecular pathway in the response to prenatal ambient air pollution exposure. Modifications to histone H3 are the most-studied histone changes at present, and these changes have been associated with changes in gene expression. 10 Previous evidence has identified specific histone H3 modifications after exposure to organic chemical compounds, 11 heavy metals, [12][13][14] and, recently, traffic-related PM exposure in adults. 15 To our knowledge, no human studies have reported on prenatal air pollution exposure and its role in histone H3 modifications.
Trimethylated H3 lysine 4 (H3K4me3) is a modification typically associated with transcriptional activity. We selected H3K4me3 for this study because it has been shown to play an important role in memory and cognitive impairment. Because air pollution exposure is known to affect the brain but the underlying molecular mechanisms are poorly understood, we hypothesized that HK4me3 might play an important function here. Global levels of H3K4me3 are increased in the hippocampus during memory formation, and H3K4 methyltransferases and H3K4 demethylases have been associated with impaired cognition in neurologic disorders. 16 Methylation of H3 lysine 36 (H3K36) plays crucial roles in the regulation of a wide range of biological processes. Deregulation of H3K36 methylation is associated with disease, including cancer.
This has been shown for lung cancer, in which alterations in histone lysine methylation are associated with clinical prognosis. 17 The H3K36 demethylase KDM2A has been shown to be frequently upregulated in non-small cell lung cancer tumors and to promote tumor growth and invasiveness. 18 In this study, we measured 2 specific histone H3 modifications (ie, H3K4me3 and H3K36me3) as well as total histone H3 levels in cord blood samples collected from newborns from the Environmental Influence on Aging (ENVIRONAGE) birth cohort. To our knowledge, this is the first report studying gestational air pollution exposure and circulating levels of histones and histone modifications. We selected these PTMs as a discovery study on the potential role of histone PTMs in the adverse effects of gestational exposure to ambient air pollution.

Study Design and Population
From the ongoing population-based birth cohort study ENVIRONAGE, 609 mother-child pairs were recruited between February 2010 and January 2017 at Hospital East Limburg (Genk, Belgium) and included in the current study. Women were recruited when they arrived at the hospital for delivery and filled out study questionnaires at the maternity ward before leaving the hospital. Inclusion criteria were singleton pregnancy and the ability to fill out questionnaires in Dutch. The overall participation rate of eligible mothers was 61.0% (1080 of 1770), and we previously demonstrated that the cohort represents births in Flanders well. 19 Study approval was obtained from the ethics committees of Hospital East Limburg and Hasselt University and has been carried out according to the Declaration of Helsinki. 20 Written informed consent was obtained from the mothers before participation. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline. Data analysis was conducted from March to August 2019.
Information on maternal age, smoking behavior (mother and coresidents), ethnicity, prepregnancy body mass index (BMI, calculated as weight in kilograms divided by height in meters squared), and parity were obtained through questionnaires. Ethnicity was classified based on the native country of the neonates' grandparents as either European (Ն2 grandparents were European) or non-European (Ն3 grandparents were not European). The study questionnaire also asked mothers to provide their residential address and to indicate whether they moved during pregnancy.
If they had, they were asked to provide their former residential address. Furthermore, they provided their exact moving date to allow for accurate exposure calculation. Perinatal parameters, such as newborn sex, birth date, birth weight, and gestational age, were collected from birth records.
Information on maternal education was missing for 7 individuals, resulting in a final study population of 609 participants.

Sample Collection
Umbilical cord blood was collected directly after delivery in BD Vacutainer plastic whole blood tubes with spray-coated K2EDTA (BD). Within 20 minutes of cord blood collection, samples were centrifuged at 3200 rpm for 15 minutes to separate plasma from blood. Plasma was collected and stored in Eppendorf tubes at −80°C until analysis.

Exposure Assessment
Based on the mother's residential address, daily mean concentrations of PM with a diameter less than 2.5 μm (PM 2.5 ), nitrogen dioxide (NO 2 ), and black carbon (BC) in micrograms per cubic meter were estimated using a high-resolution spatial-temporal interpolation method (ie, kriging) 21 in combination with a dispersion model. 22,23 This interpolation method uses hourly measured PM 2.5 pollution data collected at the official fixed-site monitoring stations (34 sites for PM 2.5 , 44 for NO 2 , and 14 for BC) and land-cover data obtained from satellite images. 24 The model chain provides daily PM 2.5 , NO 2 , and BC values on a dense, irregular receptor grid by using data both from the Belgian telemetric air-quality network and emissions from point sources and line sources. In the Flemish region of Belgium, more than 80% (R 2 = 0.8) of the temporal and spatial variability was explained by this interpolation tool.
The accuracy of the model has also been demonstrated by showing that modeled PM 2.5 and BC at residence correlates with internal exposure to nano-sized BC particles measured in urine. 25 Based on daily residential air pollution levels, we averaged trimester-specific exposure during pregnancy (first trimester, date of conception until 13th week; second trimester, 14th until 26th week; and third trimester, 27th week until delivery) and exposure during the entire period of pregnancy (date of conception until delivery). When participants moved during pregnancy, we accounted for these

Analysis of Total and Modified Histone H3 Levels
EpiQuik Global Tri-Methyl Histone Quantification Kits (Epigentek) were used to analyze and measure

Statistical Analysis
We used SAS software version 9.4 (SAS Institute) for statistical analysis. The quantities of H3K4me3, H3K36me3, and total histone H3 were log-transformed because of their nonnormal distribution. The collected data are presented as categorical data with numbers and percentages and as continuous data with means and SDs. For histone methylation levels, geometric means and 25th and 75th percentiles are given. The association of levels of total histone H3 in cord blood with prenatal air pollution exposure was assessed using a linear regression model, while accounting for the following covariates: newborn sex, ethnicity (European or non-European), gestational age (in weeks), season of delivery (winter, spring, summer, or autumn), trimester-specific apparent temperature, maternal age (in years), smoking status (never smoked, formerly smoked, or currently smokes), educational status (coded as low, no diploma or primary school; middle, high school; and high, college or university degree), prepregnancy BMI, and parity (1, 2 or Ն3). In a sensitivity analysis, we additionally adjusted for white blood cell count and the percentage of neutrophils. As data on blood cell count was missing for 116 individuals, the sensitivity analysis was performed with the remaining 493 individuals. Statistical significance was set at P < .05, and all tests were 2-tailed.  (Figure 1).

BC Exposure
We found that H3K4me3 levels were significantly associated with second and third trimester as well  (Figure 1).

Sensitivity Analysis
Because we did not know the exact cellular composition of our samples and this might affect our results, we performed an additional analysis in which we corrected for white blood cell counts and percentage of neutrophils in the samples. Although blood cell counts were missing for 116 samples, the characteristics of the population did not change (data not shown). Results were similar to our main analysis (Figure 2).

Discussion
Epigenetic modifications, such as histone modifications, are involved in mediating how early life environment affects later health. Intracellular histones are known to play an important role in the regulation of transcription and gene expression, 26 whereas extracellular histones play an important role in inflammation. Extracellular histones induce apoptosis in several human cell types. 27,28 It has been demonstrated that severely damaged tissues release high amounts of nucleosomes and free histones into the bloodstream, aggravating the clinical features of a trauma. Circulating histones act as mediators for distant organ damage (ie, lungs) through interaction with membrane phospholipids, causing cellular calcium influx. 26 Histones released in the bloodstream were found in patients with severe blunt trauma, pancreatitis, and sepsis. 6 Based on their pathological properties, extracellular nucleosomes and histones are considered pathogen-associated molecular patterns and damageassociated molecular patterns. These molecules are able to activate the innate immune system through toll-like receptors. 29 Once activated by toll-like receptors, endothelial cells were found to secrete mediators including cytokines, chemokines, reactive oxygen species, and nitric oxide. 30 The most important intracellular function of histones is to construct nucleosomes, which form the basic structure of chromatin. Changes in the structure and function of chromatin affect gene transcription and expression through fine-tuned mechanisms. Covalent modifications of the  N-terminal tail of core histones are important regulators of chromatin status, structure, and gene expression. Methylation of histone lysine can either silence or activate gene transcription, depending on the target residue. 31 Histone modification levels can predict levels of gene expression, and actively transcribed genes are characterized by high levels of H3K4me3 in the promoter region. 32 We do not know whether the cord blood circulating histones we measured are freely circulating or encapsulated in extracellular vesicles, although it is known that extracellular vesicles can capsulate histones and nucleosomes. 33 In future research, it would be interesting to extract extracellular vesicles from the cord blood plasma directly and determine whether the histones are indeed found in circulating in extracellular vesicles or rather free circulating.
To our knowledge, this is the first report investigating the association of circulating trimethylated and total histone H3 levels early in life with gestational particulate matter exposure.
Our findings showed a positive association between air pollution exposure and total histone H3 levels. This is in accordance with the observation that levels of circulating histones are increased in inflammation and sepsis 6 and that circulating histone levels are associated with acute lung injury after trauma. In mice, increased levels of circulating histones lead to an increase in the size and severity of stroke. 26 Our results are in agreement with cohort studies on environmental exposure measured in peripheral blood samples, including arsenite 34 and nickel. 35 Although it is known that H3K4me3 is associated with DNA transcription activation, the exact mechanism behind its association with environmental exposure is poorly understood. The inverse association we observed between H3K36me3 and pollution exposure corresponds to results from a study among truck drivers in Beijing, in which 14-day average ambient PM with a diameter less than 10 μm exposure was significantly and inversely associated with H3K36me3 levels in plasma leukocytes from adults. 15 It appears that both short-and long-term air pollution exposure affect H3 methylation levels, given that the study by Zheng et al 15 considered 14-day moving averages for PM exposure, whereas we observed significant associations between trimester and entire pregnancy exposure and H3 trimethylation marks. All the previously mentioned studies have measured cellular levels of histones and histone modifications, whereas our study is the first, to our knowledge, to report circulating histone levels in association with air pollution exposure.
The public health significance of our findings is currently not known. Air pollution exposure is known to affect child health, considering that it has been associated with a decrease in birth weight 36 and childhood blood pressure. 37 However, insults to the genome in the perinatal period can contribute to carcinogenesis during the life course and may be more important relative to other life stages because of the higher probability that mutated and genomically unstable cells could populate the rapidly growing tissues of an infant. 38,39 Exposure to traffic-related air pollution was shown to increase the risk of pediatric acute leukemia in the Italian Studio Epidemiologico sui Tumori Infantili Linfoemopoietici (SETIL) study. 40 Therefore, in utero exposures affecting histone (methyl) levels are potentially important in creating an adverse environment that enables carcinogenesis in later life.

Limitations
The current findings must be interpreted in the context of their limitations. At this time, we cannot make any predictions on the role of levels of trimethylated and total histone H3 in cord blood in postnatal child health or adult disease risk. Furthermore, as our analyses were performed in plasma extracted from cord blood, we do not know which cell types were responsible for the presence of circulating histones in our samples; further research focusing on which cell types drove our observations is warranted. Furthermore, although our results were consistent after multiple adjustments, we cannot exclude the possibility of residual confounding by some unknown factor that is associated with both cord blood histone H3 levels and air pollution exposure.
A potential source of bias for studies on air pollution exposure is exposure misclassification.
Errors in the measurement of PM by monitoring stations and interpolation methods used to estimate individuals' PM exposure may be a potential source of information bias. Personal PM exposure might be quite different from the estimated outdoor PM exposure since participants may spend a large